Jan W.M. Visser

A NOVEL MURINE CATHELIN-LIKE PROTEIN EXPRESSED IN BONE MARROW

A novel cDNA encoding a putative secreted protein was isolated from murine bone marrow. The encoded protein named MCLP ( urine athelin‐ ike rotein) was found to be highly homologous to the pig cathelin, and to four neutrophil antimicrobial polypeptides: CAP 18, indolicidin, Bac 5 and FALL‐39. Secondary structure prediction studies identified a highly cationic region in the C‐terminal part of prepro‐MCLP with a tendency to adopt an amphipathic α‐helical conformation, as observed in many antimicrobial peptides. However, no antibacterial activity was observed with the synthetic peptide corresponding to this region of MCLP.

Physical Separation of the Cycling and Noncycling Compartments of Murine Hemopoietic Stem Cells

Till and McCulloch (22) have demonstrated that mouse hemopoietic tissue contains a class of cells capable of giving rise to macroscopic colonies in the spleens of radiated mice. These colonies have been shown to be derived from single cells (3), the “colony-forming cells,” which are capable of extensive proliferation (3, 22), of differentiation (12, 30), and of self-renewal (23, 24). These findings support the hypothesis that blood-forming tissue contains a pluripotent, hemopoietic stem cell which has the capacity to give rise to all blood cells, including copies of itself. Pluripotent stem cells then give rise to colonies in the spleens of radiated mice. Since only a portion of the injected stem cells reach the spleen and form colonies there, the cells that form colonies have been termed colony-forming units in the spleen, or CFU-s. The number of CFU-s is then directly proportional to the number of stem cells. The possibility of quantifying the stem cell has led to the initiation of many experiments with the aim of further characterizing it.

A mass spectrometry-based approach to host cell protein identification and its application in a comparability exercise.

Host cell proteins (HCPs) are process-related impurities present in biopharmaceuticals and are generally considered to be critical quality attributes. Changes in a biopharmaceutical production process may result in qualitative shifts in the HCP population. These shifts are not necessarily detectable when overall HCP levels are measured with traditional approaches such as enzyme-linked immunosorbent assays (ELISAs). Thus, the development of techniques that complement the ELISAs functionality is desirable. Here, a mass spectrometry (MS)-based approach for the analysis of HCP populations in biopharmaceuticals is presented. It consists of (i) the generation of exclusion lists that represent the masses of the active pharmaceutical ingredient (API), (ii) the compilation of inclusion lists based on an HCP catalog derived from the analysis of protein A-purified samples, and (iii) the analysis of purified biopharmaceuticals using the generated exclusion and inclusion lists. With this approach, it was possible to increase sensitivity for HCP detection compared with a standard liquid chromatography tandem MS (LC-MS/MS) run. The workflow was successfully implemented in a comparability exercise assessing HCP populations in drug substance samples before and after a process change. Furthermore, the results suggest that size can be an important factor in the copurification of HCPs and API.

Novel CDC2-related protein kinases produced in murine hematopoietic stem cells

The polymerase chain reaction with degenerate primers was used for the amplification of cDNA encoding CDC2-related protein kinase (PK) sequences from murine hematopoietic stem cells. In total, nine different PK-encoding sequences were obtained. At least four of them encode previously unknown PKs.

Identification and purification of murine hematopoietic stem cells by flow cytometry.

Publisher Summary All the different blood cell types are derived from a common ancestor, the pluripotent hematopoietic stem cell (PHSC). This cell not only has the capacity to differentiate into committed progenitor cells of all the various blood cell lineages but also can renew itself, thus maintaining the hematopoietic organ at a steady-state level throughout life. The production of mature blood cells out of stem cells and committed progenitors requires >10 cell divisions. As a consequence, the stem cells are rare cells, even in hematopoietic organs. Stem cells are detected by the examination of their offspring after in vitro culture or after transplantation into often lethally irradiated syngeneic or congenic recipients. There exists no consensus about the different culture and in vivo transplantation methods with respect to their specificity for pluripotent stem cells. The quantitation of stem cells by those methods may lead to widely different results. Identification of PHSC by flow cytometry (FCM) should be combined with cell culture and transplantation assays for verification. Therefore, FCM methods should employ supravital staining and labeling, which can be combined with sorting, cell culture, and transplantation procedures. This chapter describes various protocols for identifying and sorting spleen colony forming. Most of these employed FCM in combination with other physical and immunological cell separation techniques have served to remove the bulk of mature cells.

Biotinylation of interleukin-2 (IL-2) for flow cytometric analysis of IL-2 receptor expression. Comparison of different methods.

The main prerequisites for the use of biotinylated ligands to study the expression of growth factor receptors on heterogeneous cell populations, such as peripheral blood or bone marrow, by flow cytometric methods, are that the biotinylated ligand retains its binding ability and that binding of the biotinylated ligand to the receptor does not inhibit the subsequent interaction of biotin with fluorescently tagged avidin or streptavidin. Using interleukin-2 (IL-2), we compared the usefulness of various biotinylation reagents, NHS-biotin, S-NHS-biotin, S-NHS-LC-biotin, DBB and photobiotin, and developed optimal biotinylation conditions for the preparation of biologically active biotin-labeled IL-2 and the detection of IL-2 receptor expressing cells by flow cytometry. As determined by spot blot analysis, biotinylation of IL-2 was most efficient at the highest biotin-to-protein (B:P) ratio used. At a B:P ratio of 100, most of the biological activity of IL-2 was retained when S-NHS-LC-biotin was used. In contrast, most of the biological activity of IL-2 samples that were labeled with NHS-biotin or photobiotin was lost under these conditions. Biotin-labeled IL-2 preparations were tested in order to detect IL-2 receptors on IL-2 dependent CTLL-2 cells by flow cytometry after sequential staining with the biotinylated IL-2 and fluorescence tagged streptavidin. A high B:P ratio generally resulted in a high specific fluorescence intensity of the cells, particularly when S-NHS-LC-biotin was used as the biotinylation reagent. Biotin-IL-2 could also be used to detect IL-2 receptors expressed by lymphocytes in peripheral blood and bone marrow. Comparison of staining of lymphocytes with biotinylated IL-2 and an antibody against the IL-2 receptor alpha chain demonstrated that only a subset of the cells that showed a strong fluorescence signal after staining with biotinylated IL-2 expressed high numbers of the IL-2 receptor alpha chain. This is in agreement with the expression of functional IL-2 receptors on resting T cells and NK cells which do not express the alpha chain. After stimulation with PHA, virtually all lymphocytes expressed the alpha chain, whereas only part of these cells showed a strong fluorescence signal after staining with biotin-IL-2, while the rest of the cells had very low numbers of IL-2 binding sites. Our results demonstrate that, in addition to staining individual receptor subunits with antibodies, staining with biotinylated IL-2 is a useful indicator of functional IL-2 receptor expression.

Separation of spleen colony-forming units and prothymocytes by use of a monoclonal antibody detecting an H-2K determinant

The density of H-2K antigens was determined on both the mouse hemopoietic stem cell, using an assay for spleen colony-forming units (CFU-S), and the prothymocyte, using a thymus repopulation assay. This was done by light-activated cell sorting of bone marrow cells labeled first with a biotinylated antibody against H-2Kk and then with avidin-fluorescein isothiocyanate. Almost all CFU-S were found to be present among the 4% bone marrow cells with high forward light scatter (FLS), low perpendicular light scatter (PLS), and bright immunofluorescence. Thymus regeneration by this brightly fluorescent fraction was delayed 3 days compared to thymus regeneration by unsorted cells, although the same number of CFU-S was present in each cell suspension. This delay indicates that differentiation from CFU-S to prothymocytes takes 3 days. The fraction of cells in the FLS/PLS window with dull anti-H-2Kk fluorescence contained few CFU-S and gave rise to a transient thymus regeneration. These findings indicate that the prothymocyte carries fewer H-2K antigens than does the CFU-S. The H-2K antigen is a marker with which CFU-S and prothymocytes can be separated. Therefore, during early T-cell differentiation, the number of H-2K molecules on the cell surface decreases (CFU-S----prothymocyte----cortical thymocyte). During maturation of T cells, a reexpression of H-2K molecules occurs, since lymph node cells and spleen cells were shown to be brightly positive for H-2K antigen.

MICE LACKING A FUNCTIONAL CHK GENE HAVE NO APPARENT DEFECTS IN THE HEMATOPOIETIC SYSTEM.

Non‐receptor tyrosine kinase Chk has been implicated in hematopoietic development. To study the function of Chk in vivo, we have generated chk‐deficient mice using gene targeting. Overall development of mice homozygous for this mutation was apparently normal. Blood counts, FACS analysis of hematopoietic cell populations, CFU‐C and CAFC assays showed no significant difference between wild type and mutant animals. Thus, the dispensability of Chk for mouse development and hematopoiesis suggests that its function may be redundant in vivo, and most likely be compensated by activity of a closely related protein tyrosine kinase Csk.

Jedi--a novel transmembrane protein expressed in early hematopoietic cells.

Self‐renewal and differentiation of hematopoietic stem and progenitor cells are defined by the ensembles of genes expressed by these cells. Here we report identification of a novel gene named Jedi, which is expressed predominantly in short‐ and long‐term repopulating stem cells when compared to more mature bone marrow progenitors. Jedi mRNA encodes a transmembrane protein that contains multiple EGF‐like repeats. Jedi and two earlier reported proteins, MEGF10 and MEGF11, share a substantial homology and are likely to represent a novel protein family. Studies of the potential role of Jedi in hematopoietic regulation demonstrated that the retrovirally mediated expression of Jedi in bone marrow cells decreased the number of myeloid progenitors in in vitro clonogenic assays. In addition, expression of Jedi in NIH 3T3 fibroblasts resulted in a decreased number of late and early myeloid progenitors in the non‐adherent co‐cultured bone marrow cells. Jedi shares a number of structural features with the Jagged/Serrate/Delta family of Notch ligands, and our experiments indicate that the extracellular domain of Jedi, similar to the corresponding domain of Jagged1, inhibits Notch signaling. On the basis of obtained results, we suggest that Jedi is involved in the fine regulation of the early stages of hematopoietic differentiation, presumably through the Notch signaling pathway. J. Cell. Biochem. 101: 767–784, 2007.

Identification of CFU-s by Scatter Measurements on a Light Activated Cell Sorter

The morphological description of the hemopoietic stem cell [the colony forming unit-spleen (CFU-s) cell] is made more difficult by the low incidence of this cell in hemopoietic tissue. An unequivocal description is only achieved if it is followed by proof that the cell described can regenerate the hemopoietic organs of irradiated mice (CFU-s assay). Therefore, maintenance of cell viability is essential. This precludes the use of the microscope as the sole instrument of investigation.